Reduced dioxygen concentration in the tissues of multicellular organisms triggers the hypoxic response that works to restore normoxia by improving the supply of oxygen to affected tissues. The response involves an array of genes including those encoding for erythropoietin and vascular endothelial growth factor and is mediated by an αβ-heterodimeric transcription factor, hypoxia-inducible factor (HIF), the α-subunit of which is upregulated under hypoxic conditions. The genes involved in the hypoxic response include those involved in angiogenesis. Modulation of the hypoxic response is of interest from the perspectives of developing new therapies for both cancer, cardiovascular and other diseases.
The super-family of 2-OG and ferrous iron dependent enzymes catalyse a wide range of oxidative reactions including the hydroxylation of unactivated C—H bonds (such as in the conversion of proline to 4-hydroxyproline as catalysed by proline-4-hydroxylase), desaturation of C—C bonds and oxidative cyclisations.
In most cases enzymes belonging to the super-family of 2-OG oxygenases (as defined by their structural relationship, requirement for dioxygen as cosubstrate, and a requirement for ferrous iron as a cofactor) actually use 2-OG as a cosubstrate. In these cases the 4-electron oxidising power of a dioxygen molecule is coupled to the two-electron oxidation of the substrate (e.g. proline to 4-hydroxyproline in reactions catalysed by proline-4-hydroxylase) and the oxidation of 2-OG to give succinate and CO2 (Que Nat. Struct. Biol. (2000) 7 182-184).
The stoichiometry of a typical hydroxylation reaction as catalysed by a 2-OG oxygenase, such as FIH or a PHD enzyme, is as follows:
      20    ⁢                  ⁢    G    +      substrate          (              eg        .                                  ⁢        proline            )        ⁢          +            O      2        ⁢                                            ⁢                              →                                                                                                                          ⁢    succinate    +      CO    2    ⁢          +      product          (              eg        .                                  ⁢        hydroxyproline            )      
Certain 2-OG oxygenases, including the prolyl hydroxylase domain (PHD also known as EGLN and HPH) enzymes, PHD1, PHD2, PHD3, and factor inhibiting hypoxia-inducible factor (FIH), are current targets for medicinal chemistry. Inhibition of the PHDs, and of FIH, is recognised as a means of inducing the hypoxic response for use in a therapeutic manner.
The sequence of FIH and its crystal structure coupled to bioinformatic analyses has identified FIH (i) as a 2-OG oxygenase that contains a double stranded beta-helix or jelly roll structural element, (ii) that FIH is a member of the so called JmjC family of proteins. The JmjC proteins are related to the cupin family as each contain, or are predicted to contain, at least one jelly roll structural element. Some of the JmjC proteins have been identified as being involved in important biological processes or disease states, e.g. congenital heart disease. It has been proposed that many of the JmjC proteins are 2-OG oxygenases involved in transcriptional regulation. A problem in the field is defining the substrates for the 2-OG oxygenases, some of which are characterised as JmjC proteins, which are involved in transcriptional regulation and other signalling pathways.
Currently there are over 400 JmjC domain proteins in the SMART domain database (Shultz et al. PNAS (1998) 95 5857-5864). A number of these are recognised as being gene products that are involved in regulation of chromatin structure and hence transcriptional control (Clissold and Ponting TIBS (2001) 26 7-9).
The ankyrin (ANK) repeat motif is composed of two anti-parallel α-helices followed by a beta-bulge and beta-hairpin containing loop connecting it to the next repeat, each of which contain 33 residues (FIG. 1). The repeats occur in tandem from several up to 24 repeats (for review see Sedgwick and Smerdon TIBS (1999) 24 311-316). The extended beta-hairpin containing loops, or “fingers”, form a groove on the surface.
Currently, over 3500 sequences containing ANK can be found listed in the SMART domain database (Shultz et al. PNAS (1998) 95 5857-5864). Of these 3500 sequences, over 3000 are from eukaryotes, 135 from bacteria, and 4 from archaea. Of the 3000 eukaryotic ANK-protein sequences, over 2600 are from metazoans and over 600 each from human and mouse. Many ankyrin proteins are also present in plants where they are involved in regulation and signaling.
Proteins containing ANK repeats are often involved in protein-protein interactions. The highly conserved core serves as a scaffold for the variable surface exposed residues especially in the “fingers”, which are involved in most interactions with other proteins (Sedgwick and Smerdon TIBS (1999) 24 311-316). Their functions vary widely and include cyclin-dependent kinase (CDK) inhibitors, transcriptional regulators, cytoskeletal organizers, developmental regulators and even toxins. Defects in ankyrin repeat proteins have been found in a number of human diseases.